CN114574482A - Huashan new wheat straw whole genome liquid phase chip and application - Google Patents
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Abstract
The invention belongs to the technical field of genome-wide gene chips, and relates to a Huashan new wheat straw genome-wide liquid phase chip and application thereof. The liquid phase chip comprises an independently packaged Ryegrass 80K probe mixed solution and a hybridization capture reagent, wherein the Ryegrass 80K probe mixed solution comprises Ryegrass genome 49K oligonucleotide probe sites, and the total amount of the Ryegrass 80K probe sites is 80K liquid phase target capture probes. The liquid phase chip can rapidly and accurately identify the chromosome and the gene composition of exogenous ryegrass from wheat genetic background, and is effectively applied to identification, gene analysis and the like of ryegrass from Huashan and wheat families.
Description
Technical Field
The invention belongs to the technical field of genome-wide gene chips, relates to a Huashan New wheat straw genome-wide liquid phase chip and application thereof, and particularly relates to a Huashan New wheat straw genome-wide liquid phase chip developed based on a targeted sequencing technology and application of the liquid phase chip in research and development aspects such as Huashan New wheat straw variety identification and analysis.
Background
The Huashan new wheat straw (Psathysostachys hushanica Keng, 2n 2x 14, NsNs) belongs to the wheat family (Triticeae) new wheat straw (Psathysostachys) belonging to Poaceae, is a perennial diploid cross-pollinated herbaceous plant, is listed as a first rare protective plant (preferential protective species) in China and a crop wild kindred species which needs to be protected urgently, has a plurality of excellent properties such as disease resistance (stripe rust, powdery mildew, gibberellic disease and the like), cold resistance, drought resistance, saline alkali resistance, early ripening and the like, and is an important kindred species for genetic improvement of wheat. At present, a series of wheat-Huashan New wheat straw alloaddition line, allosubstitution line, translocation line, introgression line and other materials are created and identified by utilizing distant hybridization and backcross of wheat and Huashan New wheat straw by a plurality of researchers, and the wheat-Huashan New wheat straw alloaddition line, the allosubstitution line, the translocation line, the introgression line and other materials have excellent properties such as disease resistance, stress resistance and the like which are superior to those of parents. The existing wheat variety Wen nong 501 is a national wheat variety (national wheat 20200020, variety right: 20161795.5.1) successfully cultivated by utilizing a wheat-Huashan New wheat grass derived line H8-4, and has the characteristics of disease resistance and high yield.
In the process of transferring genetic materials of the ryegrass from the ryegrass in the mountains to the wheat, the accurate identification and tracking of exogenous materials of the ryegrass in the mountains are very important. In the early days, species-specific molecular markers were developed primarily to identify wheat background Lolium huashanense genomes. For example, in 2013, patent CN201310171848.6 discloses a pair of huashanxinwheatgrass specific scarr markers, which can be used for identifying wheat-huashanxinwheatgrass complete set of alien addition line materials; in 2015, a patent CN201510470522.2 discloses a pair of species-specific SCAR (specific sequence amplification) markers of ryegrass of Huashan mountain, which can quickly identify whether the distant hybrid material of wheat and ryegrass of Huashan mountain contains the bloody border of ryegrass of Huashan mountain; in 2018, patent CN201810784350.X utilizes gene database information to develop 34 SSR and 102 EST molecular specific marker primer pairs of ryegrass from Huashan mountain, and significant differences exist among ryegrass from Huashan mountain, wheat, part of homologous groups and exogenous materials.
In conclusion, researchers can provide reference for tracking genetic materials of ryegrass of Huashan in wheat background by identifying the specific marker of ryegrass of Huashan, but because the molecular marker is very limited, the proportion of the ryegrass of Huashan covered on genome or gene is too small, and the basic research and the application in the genetic improvement of wheat of the ryegrass of Huashan are greatly limited.
Disclosure of Invention
The invention aims to provide a genome-wide liquid chip of ryegrass from Huashan and application thereof, wherein the liquid chip developed based on a targeted capture technology comprises an 80K ryegrass from Huashan and a liquid-phase targeted capture probe, so that genetic substances of the ryegrass from Huashan can be quickly and effectively tracked, the transfer and utilization of excellent properties of the ryegrass from Huashan to receptor varieties can be accelerated, the breeding process can be accelerated, and the breeding efficiency of new varieties can be improved.
The technical solution of the present invention to solve the above technical problems is as follows.
A Huashan Xin Lolium Whole genome liquid chip, Huashan Xin Lolium Whole genome liquid chip is formed by 80K probe mixed solution and hybridization capture reagent of Huashan Xin Lolium packaged independently; the 80K probe mixed solution of the ryegrass in Huashan comprises 49K oligonucleotide probe sites of the whole genome of the ryegrass in Huashan, and the total 80K liquid phase target capture probes.
Further, the core site information of the 80K liquid phase targeted capture probe is shown in table 1.
Furthermore, the design process of the targeted capture probe of the lolium huashanense whole genome liquid phase chip is that the length of the probe is 110bp, the GC content of the probe is between 30% and 80%, the number of homologous regions is less than or equal to 5, the selected region is a specific sequence region of the lolium huashanense compared with wheat species, and the priority is as follows: combining the gene region > gene promoter region (2Kb) > gene downstream adjacent region (2Kb) > gene spacer region with the principle of uniform distribution of chromosomes to obtain the ryegrass 80K liquid phase targeted capture probe.
Furthermore, in the Huashan new wheat straw whole genome liquid phase chip, a liquid phase target capture probe carries out single-chain nucleotide synthesis according to a designed nucleotide sequence, and two synthesized DNA nucleotide sequences with the length of 110bp and the 5' end modified by a biotin group are called Huashan new wheat straw liquid phase probes; and (3) mixing the synthesized ryegrass huashanensis liquid phase probe in an equimolar mass, and carrying out constant volume of 3pmol/mL by using EDTA and Tris-HCl mixed solution to obtain a ryegrass huashanensis whole genome liquid phase chip probe mixed solution.
Furthermore, the Huashan new wheat straw whole genome liquid phase chip is a target capture probe and is coupled on fluorescent microspheres through a C12 molecular arm and amino modification, and each fluorescent microsphere is coupled with one target capture probe.
A Huashan Xin Lolium whole genome liquid phase chip can be used for identifying the material of the alien addition line, alien substitution line, translocation line and introgression line generated by wheat-Huashan Xin Lolium and the derived offspring of wheat and other Ns genome species of wheat family.
Furthermore, the Huashan new wheat straw whole genome liquid phase chip can be used for analyzing the chromosome composition, the chromosome structure and the homologous relation of the genome species of other Ns of the neobarley grass family and the wheat family.
Furthermore, the Huashan New wheat straw whole genome liquid phase chip can be used for genetic diversity analysis, variety identification, genetic relationship identification and whole genome association analysis of Huashan new wheat straw, wheat family other Ns genome species and derivative materials thereof.
The Huashan new wheat straw whole genome liquid phase chip and the application thereof have the beneficial effects that:
(1) the liquid phase chip developed based on the targeted capture technology comprises a group of 80K liquid phase targeted capture probes for the complete genome of the ryegrass of Huashan mountain;
(2) the liquid phase chip can be applied to the identification of materials formed by distant hybridization of wheat and ryegrass of Huashan mountain, and the genome composition and exogenous fragment gene carrying condition of ryegrass of Huashan mountain in various derivative materials can be directly obtained by applying the chip, so that the rapid identification of the ryegrass of Huashan mountain distant hybridization materials is realized;
(3) the liquid phase chip can be applied to the derived material and variety potential exogenous fragment formed by distant hybridization of the Huashan new wheat straw and the wheat, exogenous gene analysis and cloning, accurately locks the gene composition of the derived material and variety potential exogenous fragment, and assists in gene cloning;
(4) the liquid phase chip captures 35884 gene regions or adjacent region sequences of the genome of the ryegrass of Huashan with a target direction, and can be applied to genetic diversity analysis, variety identification, genetic relationship identification, DNA fingerprint database construction and genome-wide association analysis of the ryegrass of Huashan and derived materials thereof;
(5) the liquid phase chip can rapidly and accurately identify the chromosome and the gene composition of the exogenous Xin Huashan wheat Ns group under the genetic background of wheat, and is effectively applied to genetic diversity analysis, variety identification, genetic relationship identification and whole genome association analysis of the Xin Huashan wheat and other Ns genome species derived materials of wheat families;
(6) the chip is a liquid-phase chip, compared with a solid-phase chip, the liquid-phase chip has high detection accuracy, more effective information and high flexibility and diversity, effectively reduces the detection cost, and provides important technical support for utilizing the Huashan new wheat straw to improve the wheat heredity.
Drawings
FIG. 1 is a genome distribution diagram of the Huashan New wheat straw whole genome liquid phase chip of the present invention;
FIG. 2 is a diagram showing the distribution of the capture efficiency of the target sites of the liquid phase chip of Ryegrass on Huashan mountain.
Detailed Description
Example 1
A Huashan Xin Lolium Whole genome liquid phase chip is composed of an independently packaged Huashan Xin Lolium 80K probe mixed solution and a hybridization capture reagent.
The 80K probe mixed solution of the ryegrass in Huashan comprises 49K oligonucleotide probe sites of the whole genome of the ryegrass in Huashan, and the total 80K liquid phase target capture probes. Specifically, the Huashan New wheat grass whole genome liquid phase chip is formed by mixing 49090(49K) Huashan new wheat grass compared with wheat species specific oligonucleotide sites, and 80391(80K) liquid phase target capture probes are counted; of these, about 90.8% of the probes were located in the gene region or gene neighborhood (2Kb upstream and downstream), and involved 35884 genes in the entire genome of Lolium huashanense.
The hybridization capture reagent is a GenoBaits DNA-seq Library Prep kit from Boyle biotechnology, Inc. of Shijiazhuang Boylei, and comprises independently packaged GenoBaits Block I, GenoBaits Block II, GenoBaits 2 × Hyb Buffer, GenoBaits Hyb Buffer Enhancer, GenoBaits 2 × Beads Wash Buffer, GenoBaits 10 × Wash Buffer I, GenoBaits 10X Wash Buffer II, GenoBaits 10X Wash Buffer III and GenoBaits 10X Stringent Wash Buffer.
Wherein, the gene distribution of the 80K liquid phase target capture probe site is shown in figure 1, and the core site information is shown in table 1.
TABLE 1.80 core site information for liquid phase targeted capture probe sites
In table 1, the site information is specifically: the left 1 Ns-7 Ns in the locus number represent the chromosome where the locus is located, and the subsequent two groups of numerical values are respectively the corresponding start and stop sites of the oligonucleotide probe on each chromosome of the Lolium huashanense REFERENCE genome (Psathysostachys _ hushanica-REFERENCE-NWSUAF-1.0).
Example 2
Example 1 screening of a 80K liquid phase targeted capture probe for the genome of ryegrass from huashanensis in a specific method is as follows:
(1) downloading re-sequencing sequences of spring wheat (CNCB database accession number CRR062130) and Abbo (CNCB database accession number CRR062102) varieties, performing sequence comparison with a ryegrass REFERENCE genome (Psathysostachys _ hushanica-REFERENCE-NWSUAF-1.0) of the ryegrass of Huashan by using BWA software, and extracting an unordered coverage area of the ryegrass genome of Huashan based on the sequence comparison, namely a ryegrass of Huashan compared with a potential species specificity interval of wheat;
(2) according to the information of the initial and termination positions of the potential Huashan new wheat straw species specific interval, taking an average value, and based on the average value, extending 150bp extraction sequences left and right, namely representing 300bp sequences of the interval;
(3) using a gmap software and a BLAST software to carry out sequence alignment on the gene sequence and a wheat reference Genome (Ensemble Genome database: Triticum _ aestivum.IWGSC.41), and further removing alignment sites capable of being aligned with the wheat Genome; comparing the reserved sites with a ryegrass reference genome in Huashan to remove a multi-site comparison sequence;
(4) performing targeted capture probe evaluation on the candidate sequences in each region to complete the design of the liquid chip probes, wherein 2 liquid chip capture probes are arranged at each site;
(5) according to the probe sites of the designed liquid phase chip, combining the annotation information of the genome reference of the ryegrass from Huashan, the probes are screened according to the following priority standard: the gene region > gene promoter region (2Kb) > gene downstream adjacent region (2Kb) > gene spacer region, and the 49K oligonucleotide sites with uniformly distributed ryegrass chromosomes in the Huashan are finally obtained by combining the principle of uniform distribution of chromosomes, and the corresponding 80K liquid phase chip directional capture probes are obtained.
And (3) carrying out probe synthesis on the obtained ryegrass huashanensis 80K liquid phase targeted capture probe by the method, thereby obtaining the ryegrass huashanensis whole genome liquid phase chip.
Example 3
The Huashan new wheat straw liquid phase chip is developed based on a targeted capture technology, and the working principle is that based on the design of a probe and the DNA of a detection sample, targeted capture and sequencing are carried out through base complementary pairing, so that the purposes of target area gene detection and typing are realized; specifically, the design process of the liquid phase targeted capture probe of the lolium huashanense whole genome liquid phase chip is that the length of the probe is 110bp, the GC content of the probe is 30-80%, the number of homologous regions is less than or equal to 5, the selected region is a specific sequence region of the lolium huashanense compared with wheat species, and the priority is as follows: the gene region > gene promoter region (2Kb) > gene downstream adjacent region (2Kb) > gene spacer region, and the 80K liquid phase target capture probe of the ryegrass from Huashan is obtained by combining the principle of uniform distribution of chromosomes.
The liquid phase target capture probe carries out single-chain nucleotide synthesis according to the designed nucleotide sequence, and two synthesized DNA nucleotide sequences with the length of 110bp and the 5' end modified by a biotin group are called as a Huashan new wheat straw liquid phase probe; and (3) mixing the synthesized ryegrass huashanensis liquid phase probe in an equimolar mass, and carrying out constant volume of 3pmol/mL by using a mixed solution of EDTA and Tris HCl to obtain a ryegrass huashanensis whole genome liquid phase chip probe mixed solution.
The Huashan new wheat straw whole genome liquid phase chip is a target capture probe which is coupled on fluorescent microspheres through a C12 molecular arm and amino modification, and each fluorescent microsphere is coupled with one target capture probe.
Example 4
The genome genetic information of the ryegrass from Huashan can be efficiently captured by the genome whole liquid phase chip from the ryegrass from Huashan obtained in the above embodiment, and the specific implementation process is as follows.
(1) Extracting and quality testing of DNA of a material to be tested: taking the Xin wheat straw from the mountain to naturally dry leaves, and extracting DNA by using a CTAB method; detecting the DNA of the sample by using agarose gel electrophoresis to ensure the integrity of the genome; and (3) measuring the concentration of the genomic DNA by using a micro ultraviolet spectrophotometer, and adjusting the working concentration of the DNA of each sample to 10-50 ng/ul.
(2) Detection of Huashan New wheat straw whole genome liquid chip: the liquid-phase chip DNA hybrid capture library was constructed according to the standard protocol of the targeted gene capture Technology for liquid-phase probe hybridization (http:// www.molbreeding.com/index. php/Technology/GenoBaits. html), and library sequencing was dependent on the Wash MGISEQ2000 sequencing platform.
(3) And (3) data analysis: the offline sequence utilizes FastQC software to carry out quality control to obtain Clean data; then, comparing the gene sequence with the genome of the ryegrass in Huashan by using BWA software to obtain comparison files of each sample, and counting the sequence coverage condition of each probe site by using bdtools; the detection sites are compared with the chip sites, and the result shows that the set of chips can efficiently capture the target sites (as shown in figure 2); meanwhile, based on the comparison files of all samples, SNP of all detected sites can be detected by using a standard flow of GATK software, and then genotyping is carried out on different Lolium huashanense.
The liquid phase chip can also be applied to the analysis and cloning of derived materials and variety potential exogenous fragments formed by distant hybridization of wheat, such as the cloning of excellent trait genes of the ryegrass derived materials from Huashan, and the discovery of the excellent trait regulatory genes by the traditional map-based cloning method is very difficult because of the low genetic recombination rate of exogenous genomes and wheat, and the chip set comprises 80K liquid phase probes of the ryegrass from Huashan, so that the gene composition of the derived materials and the variety potential exogenous fragments can be accurately locked, and the cloning of genes is assisted.
The liquid-phase chip captures 35884 gene regions or adjacent region sequences of the genome of the ryegrass of Huashan in a targeted manner, so that the chip can be applied to genetic diversity analysis, variety identification, genetic relationship identification, DNA fingerprint database construction and genome-wide association analysis of the ryegrass of Huashan and derived materials thereof.
The liquid phase chip can rapidly and accurately identify the chromosome and the gene composition of the exogenous Xin Huashan wheat Ns group under the genetic background of wheat, and is effectively applied to genetic diversity analysis, variety identification, genetic relationship identification and whole genome association analysis of the Xin Huashan wheat and other Ns genome species derived materials of wheat families.
Claims (8)
1. The Huashan New wheat straw whole genome liquid phase chip is characterized by comprising an independently packaged Huashan New wheat straw 80K probe mixed solution and a hybridization capture reagent, wherein the Huashan New wheat straw 80K probe mixed solution comprises Huashan New wheat straw whole genome 49K oligonucleotide probe sites, and a total of 80K liquid phase target capture probes.
2. The lolium huashanense whole genome liquid phase chip of claim 1, wherein the core site information of the 80K liquid phase target capture probe site is as follows:
wherein, the left 1 Ns-7 Ns in the locus number represent the chromosome of the locus, and the subsequent two groups of numerical values are respectively the starting and ending sites of the oligonucleotide probe corresponding to each chromosome on the Lolium huashanense reference genome.
3. The lolium huashanense whole genome liquid phase chip of claim 2, wherein the design process of the liquid phase target capture probe is that the length of the probe is 110bp, the GC content of the probe is between 30% and 80%, the number of homologous regions is less than or equal to 5, the selected region is a specific sequence region of the lolium huashanense compared with wheat species, and the priority is: the gene region > gene promoter region (2Kb) > gene downstream adjacent region (2Kb) > gene spacer region, and the 80K liquid phase target capture probe of the ryegrass from Huashan is obtained by combining the principle of uniform distribution of chromosomes.
4. The lolium huashanense whole genome liquid phase chip of claim 3, wherein the liquid phase target capture probe carries out single-stranded nucleotide synthesis according to the designed nucleotide sequence, and two synthesized DNA nucleotide sequences with the length of 110bp and the 5' end modified by a biotin group are called lolium huashanense liquid phase probe; and (3) mixing the synthesized ryegrass huashanensis liquid phase probe in an equimolar mass, and carrying out constant volume of 3pmol/mL by using EDTA and Tris-HCl mixed solution to obtain a ryegrass huashanensis whole genome liquid phase chip probe mixed solution.
5. The ryegrass genome-wide liquid phase chip of claim 3, wherein the ryegrass genome-wide liquid phase chip is a target capture probe coupled to fluorescent microspheres through a C12 molecular arm and an amino modification, and each fluorescent microsphere is coupled to one target capture probe.
6. The use of the Huashan New wheat straw whole genome liquid phase chip according to any one of claims 1 to 5, wherein the Huashan New wheat straw whole genome liquid phase chip is used for identifying the material of the alien addition line, alien substitution line, translocation line and introgression line generated by wheat-Huashan New wheat and the progeny derived from other Ns genome species of wheat family.
7. The use of the S.huashanensis genome-wide liquid chip of any one of claims 1 to 5, wherein the S.huashanensis genome-wide liquid chip is used for analyzing the chromosome composition, chromosome structure and homology of genomic species of neobarley and other Ns of the wheat family.
8. The use of the Ryegrass Huashan genome Walsh liquid chip of any one of claims 1-5, wherein the Ryegrass Huashan genome liquid chip is used for genetic diversity analysis, variety identification, genetic relationship identification, genome wide association analysis of Ryegrass Huashan, other Ns genomic species of wheat and their derivatives.
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